Co-Cultured Methanogen Improved the Metabolism in the Hydrogenosome of Anaerobic Fungus As Revealed by Gas Chromatography-Mass Spectrometry Analysis

Open Access Asian-Australas J Anim Sci Vol. 33, No. 12:1948-1956 December 2020 https://doi.org/10.5713/ajas.19.0649 pISSN 1011-2367 eISSN 1976-5517

Co-cultured methanogen improved the metabolism in the hydrogenosome of anaerobic fungus as revealed by gas chromatography-mass spectrometry analysis

Yuqi Li1, Meizhou Sun1, Yuanfei Li1, Yanfen Cheng1,*, and Weiyun Zhu1

* Corresponding Author: Yanfen Cheng Objective: The purpose of this study was to reveal the metabolic shift in the fungus co- Tel: +86-25-84395523, Fax: +86-25-84395523, E-mail: [email protected] cultured with the methanogen (Methanobrevibacter thaueri). Methods: Gas chromatography-mass spectrometry was used to investigate the metabolites 1 Laboratory of Gastrointestinal Microbiology, National in anaerobic fungal (Pecoramyces sp. F1) cells and the supernatant. Center for International Research on Animal Gut Nutrition, Nanjing Agricultural University, Nanjing Results: A total of 104 and 102 metabolites were detected in the fungal cells and the super 210095, China natant, respectively. The partial least squares-discriminant analysis showed that the metabolite profiles in both the fungal cell and the supernatant were distinctly shifted when co-cultured ORCID Yuqi Li with methanogen. Statistically, 16 and 30 metabolites were significantly (p<0.05) affected in https://orcid.org/0000-0002-1527-2039 the fungal cell and the supernatant, respectively by the co-cultured methanogen. Metabolic Meizhou Sun pathway analysis showed that co-culturing with methanogen reduced the production of https://orcid.org/0000-0002-8871-2322 Yuanfei Li lactate from pyruvate in the cytosol and increased metabolism in the hydrogenosomes of https://orcid.org/0000-0002-6712-3473 the anaerobic fungus. Citrate was accumulated in the cytosol of the fungus co-cultured with Yanfen Cheng the methanogen. https://orcid.org/0000-0001-8935-9551 Weiyun Zhu Conclusion: The co-culture of the anaerobic fungus and the methanogen is a good model https://orcid.org/0000-0001-9222-6581 for studying the microbial interaction between H2-producing and H2-utilizing micro organisms. However, metabolism in hydrogenosome needs to be further studied to gain Submitted Aug 15, 2019; Revised Nov 10, 2019; better insight in the hydrogen transfer among microorganisms. Accepted Jan 6, 2020 Keywords: Anaerobic Microorganism; Methanogenium; Cytosol; Hydrogenosomes

INTRODUCTION

Anaerobic fungi, found in the rumen, hindgut, and feces of ruminants and other herbivores, are important fiber degrading microoganisms [1]. They are assigned to the phylum Neocalli- mastigomycota and 11 genera have been described so far, namely: Neocallimastix, Caecomyces, Piromyces, Orpinomyces, Anaeromyces, Cyllamyces, Buwchfawromyces, Oontomyces, Peco ramyces, Feramyces, and Liebetanzomyces [2]. Anaerobic fungi not only produce a variety of fiber degrading enzymes, but also penetrate the plant structure by their rhizoids [3]. Previous studies showed that the fiber degrading ability of anaerobic fungi was signifi-

cantly improved by co-cultured H2-utilizing methanogens [4-6]. This improvement might be due to shift in the metabolic pathways in anaerobic fungi, which increases production of substrates for the co-cultured methanogens and thus reduces production of other metabolites. Bauchop & Mountfort cultivated an anaerobic fungus, Neocallimastix frontalis,

with H2-formate-utilizing methanogen and found that concentrations of lactate, ethanol, and formate were significantly decreased, while the concentration of acetate was significantly increased [5]. During the last decades, similar results have been reported in several studies [6-8].

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Using genomic, transcriptomic, and proteomic data, Li et fungal cells were collected according to the methods of Marvin- al investigated the shift in the metabolic pathways of anaero- Sikkema et al [11]. The bottles were centrifuged at 200 g for bic fungus Pecoramyces sp. F1 co-cultured with a syntrophic 10 min to separate the fungal cells and the supernatant. The methanogen [2]. These workers demonstrated that the ex- fungal cells were washed using 20 mM phosphate-buffered pression of many genes involved in the metabolic pathway saline (PBS) containing 20 mM dithiothreitol and centrifu- was downregulated by the syntrophic methanogen. However, gated at 200 g for 10 min. The procedure was repeated twice, only a limited number of proteins involved in the metabolic and the pellet was dissolved in 2 mL PBS. To break the cells, pathway of the anaerobic fungus were downregulated and 1 mm glass beads were added, and the mixture was vortexed no significant positive correlation was observed between the for 10 min. It was then centrifuged at 500 g for 15 min. The transcriptomic data and the proteomic data. The purpose of supernatant was collected to obtain cell contents. The fungal the study was to investigate the metabolites in an anaerobic supernatant was centrifuged at 13,000 g for 10 min before fungus (Pecoramyces sp. F1) and the metabolic shift that occurs the analysis. The samples were stored at –80°C for the gas when it is co-cultured with a methanogen (Methanobrevibacter chromatography-mass spectrometry (GC/MS) analysis. thaueri). Sample preparation and gas chromatography-mass MATERIALS AND METHODS spectrometry analysis To an aliquot of 50 μL of the cell content or the fungal su- Microorganisms and maintenance pernatant was added 200 μL methanol containing 12.5 μg/mL 13 The co-culture of anaerobic fungus and methanogen used in myristic-1,2- C2 acid as an internal standard (IS). The mixture the present study was isolated from the rumen of goat [6]. The was vortexed for 5 min, placed at 4°C for 1 h, and centri- collection procedure of rumen fluid was approved by the fuged at 20,000 g for 10 min at 4°C. An aliquot of 100 μL Animals Care and Use Committee of Nanjing Agricultural supernatant was transferred into a GC vial and evaporated University (SYXK (SU) 2017–0007). The anaerobic fungus to dryness under vacuum (Thermo Fisher Scientific, Asheville, was Pecoramyces sp. F1 [2] and the methanogen was Metha NC, USA). An aliquot of 30 μL methoxyamine in pyridine nobrevibacter thaueri [6]. The co-culture was maintained in (10 mg/mL) was added and vortexed for 3 min. After 16 h a medium containing rumen fluid with 1% (w/v) rice straw as of methoximation reaction at room temperature, 30 μL of a substrate and transferred every 3 days in the fresh medium N-methyl-N-(trimethylsilyl) trifluoroacetamide containing [8]. The culture medium contained penicillin (1,600 U/mL) 1% trimethylchlorosilane was added, vortexed for 1 min, and streptomycin (2,000 U/mL) to inhibit growth of bacteria. and placed at room temperature for 1 h for trimethylsilylation. The monoculture of the anaerobic fungus was obtained by Finally, 30 μL methyl myristate in heptane (30 μg/mL) was adding chloramphenicol at a final concentration of 50 mg/L, added and vortexed, and an aliquot of 0.5 μL was used for which inhibits growth of methanogens and bacteria in the the GC/MS analysis. co-culture [8]. The monoculture was transferred every 3 days The analysis of metabolites was conducted by a GC/MS in the same medium as used for the co-culture. system (SHIMADZU GC/MS QP2010Ultra/SE, Kyoto, Japan) fitted with a RTx-5MS capillary column (30 m×0.25 mm, Experimental design and sample collection Agilent J&W Scientific, Folsom, CA, USA). The conditions For the experiment, a modified medium M2, without rumen used for GC/MS analysis were as follows: Helium as carrier fluid, was used with 20 mM glucose as the substrate [9]. The gas at flow rate of 1.5 mL/min; initial column temperature of medium (90 mL) after adjusting pH to 6.8 was anaerobically 70°C for 3 min followed by increasing from 70°C to 300°C at dispensed into a serum bottle (180 mL capacity). The serum a rate of 20°C/min and held for 3 min; transfer line tempera- bottle with 100% CO2 headspace was sealed with butyl rubber ture of 205°C and ion source temperature of 200°C; ion source stopper and aluminum crimp to maintain anaerobic condi- voltage of –70 eV and acceleration voltage of –950 V; and tion [10]. Ten bottles were used for mono- and co-cultures, solvent delay of 300 s. The MS data were acquired over the respectively (i.e. a total of 20 bottles), with 5 replicates each range between m/z 50 – 800 [12]. for the experimental group and the control group. The experimental group was inoculated with 10 mL of the 3-day Data collection and processing old culture and the control group was inoculated with 10 mL After rejecting the peaks with signal-to-noise (S/N) lower of the blank medium. Thereafter, the bottles were incubated than 30 and normalizing with IS, the retention time, reten- at 39°C for 72 h without shaking [8]. After inoculation, the tion index, and mass spectra of each peak were obtained and air pressure in the fermentation bottle was balanced with a compared with the reference standards and database includ- pressure converter to make the initial air pressure 0. At the ing NIST library (2008), Wiley library, and in-house database end of the fermentation, samples of the supernatant and the established by China Pharmaceutical University [12,13]. The

www.ajas.info 1949 Li et al (2020) Asian-Australas J Anim Sci 33:1948-1956 partial least squares-discriminant analysis (PLS-DA) was fungus and the methanogen including 6-hydroxy-9H-purine, conducted using the R package ropls [14] and significant citric acid, threonine, glutamic acid, isoleucine, monostearin, difference between two groups was considered at p<0.05 uracil and valine. The remaining 8 metabolites were signifi- and variable importance plot (VIP)>1. Data visualization cantly decreased, including D-galactono-1,4-lactone, glucose, was done in R studio (www.rstudio.com). ribopyranose, beta-D-methylglucopyranoside, ribonic acid and three unknown metabolites. Many of these amino acids RESULTS participate in cytosol metabolism, and citric acid is an important intermediate in the oxidation pathway of the tricarboxylic Effects of the co-cultured methanogen on metabolic acid (TCA) cycle in cytosol metabolism. changes in the fungal cell The typical GC/MS chromatograms of metabolites in cells Effects of the co-cultured methanogen on metabolic of the anaerobic fungus with/without the methanogen are changes in the fungal supernatant shown in Figure 1. A total of 104 metabolites were detected The typical GC/MS chromatograms of metabolites in super- in the fungal cell and a distinct difference of the metabolite natant of the anaerobic fungus with/without the methanogen profiles between the two groups was observed in the PLS- are shown in Figure 3. A total of 102 metabolites were detect- DA score plot (Figure 2A). For evaluation of the model, the ed in the supernatant of the cultures and a distinct difference R2X, R2Y, and Q2 (cum) were calculated as 0.532, 0.997, in the metabolite profiles between the two groups was ob- and 0.886, respectively. Statistical analysis showed that 16 served in the PLS-DA score plot (Figure 4A). The R2X, R2Y, metabolites differed significantly between the two groups and Q2 (cum) of the model were 0.453, 0.99, and 0.898, re- (p<0.05) (Figure 2B). As shown in Table 1, 8 metabolites spectively. Statistical analysis showed that 30 metabolites significantly increased in the co-culture of the anaerobic differed significantly between the two groups (Figure 4B).

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Figure 1. Typical gas chromatography-mass spectrometry (GC/MS) chromatograms of metabolites in the fungal cells with/without the co-cultured methanogen. (A) Monoculture of the anaerobic fungus. (B) Co-culture of the anaerobic fungus and the methanogen. 1, lactate; 2, alanine; 3, Ua3; 4, oxalate; 5, α-aminobutyric acid; 6, 2-keto-3-methylvaleric acid; 7, valine; 8, carbamic acid; 9, leucine; 10, glycerol-3TMS; 11, isoleucine; 12, fumaric acid; 13, threonine; 14, UN01; 15, lauric acid; 16, taurine; 17, internal standard; 18, histidine; 19, glucose; 20, tyrosine; 21, palmitic acid; 22, oleic acid; 23, stearic acid; 24, UI22; 25, UI23.

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Figure 2. Effects of the co-cultured methanogen on the metabolites in the fungal cell, as evaluated by the PLS-DA analysis. (A) PLS-DA score plot. (B) Relationship between VIP and p-value. PLS-DA, partial least squares-discriminant analysis; VIP, variable importance plot.

As shown in Table 2, 22 metabolites were significantly in- Effect of the co-cultured methanogen and the anaerobic creased in the co-culture of the anaerobic fungus and the fungus on the metabolic pathway of the fungus methanogen and the remaining 8 metabolites were signifi- Based on the metabolite profiles in the cells of the anaerobic cantly decreased. fungus with/without the co-cultured methanogen and previ-

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Table 1. Significantly changed metabolites in the anaerobic fungal cells by the ous reports [2,15], the metabolic pathway of the anaerobic co-cultured methanogen fungus is shown in Figure 5. Combined with the previous Compounds Fold change (bc/ac) p-value1) VIP1) research conducted in our laboratory, including the study of 6-Hydroxy-9H-purine 2.432 0.008 1.633 the metabolic pathway differences in the utilization of glucose Citric acid 7.620 0.008 2.053 by Pecoramyces sp. F1 in monoculture and co-culture with D-Galactono-1,4-lactone 0.189 0.008 1.320 the methanogen, as well as the research on the effect of the Glucose 0.092 0.008 1.470 co-cultured methanogen on the dynamic profile of interme- Ribopyranose 0.042 0.008 1.182 diate and end metabolic pathway of Pecoramyces sp. F1, we S07 0.139 0.008 1.195 conclude that co-cultured methanogen stimulated the meta- Beta-D-Methylglucopyranoside 0.094 0.012 1.524 bolic pathway to citrate and malate in the cytosol of the Threonine 1.443 0.016 1.547 UI23 0.298 0.016 1.660 anaerobic fungus and stimulated the metabolism in its hy- Glutamic acid 1.963 0.032 1.365 drogenosome. Isoleucine 1.680 0.032 1.512 Monostearin 1.497 0.032 1.342 DISCUSSION S04 0.654 0.032 1.241 Uracil 1.869 0.032 1.480 In the recent past, several studies have focused on the effects Valine 1.870 0.032 1.462 of culturing anaerobic fungi with methanogens. For example, Ribonic acid 0.133 0.045 1.516 several workers [4-7,16] have shown higher fiber degrading bc, fungal cell of the co-culture of anaerobic fungus and methanogen; ac, fungal ability of anaerobic fungi when co-cultured with methanogens cell of the anaerobic fungal monoculture; VIP, variable importance plot. 1) When p<0.05 and VIP>1, there is a significant difference in metabolites as compared with when they were grown as a monoculture. between bc and ac. So far, approximately 60 co-cultures of anaerobic fungi and

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Figure 3. Typical gas chromatography-mass spectrometry chromatograms of metabolites in the supernatant of the anaerobic fungus with/without the co-cultured methanogen. (A) Monoculture of anaerobic fungus. (B) Co-culture of anaerobic fungus and methanogen. 1, pyruvic acid; 2, lactate; 3, alanine; 4, oxalate; 5, urea; 6, isoleucine; 7, serine; 8, threonine; 9, aminomalonic acid; 10, malic acid; 11, glutamic acid; 12, taurine; 13, glycerol-3-phosphate; 14, internal standard; 15, glucose; 16, linoleic acid; 17, cystin.

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Figure 4. Effects of co-cultured methanogen on the metabolites in the supernatant of cultures as evaluated by PLS-DA analysis. (A) PLS-DA score plot. (B) Relationship between VIP and p-value. PLS-DA, partial least squares-discriminant analysis; VIP, variable importance plot. methanogen combinations have been isolated from different one of such co-cultures, which were isolated from the rumen herbivores [6,17-20]. The co-culture of Pecoramyces sp. F1 of Chinese local goat and demonstrated to have higher fiber and Methanobrevibacter thaueri, used in the present study, is degrading ability than others [6]. The anaerobic fungus in the

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Table 2. Significantly changed metabolites in supernatant of the anaerobic co-culture was identified asPiromyces sp. F1 according to its fungus by the co-cultured methanogen morphology and renamed as Pecoramyces sp. F1 by using Compounds Fold change (bs/as) p-value1) VIP1) molecular techniques [2]. 6-Hydroxy-9H-purine 0.575 0.008 1.688 Present study showed that the metabolic pathways in the Alanine 1.334 0.008 1.692 anaerobic fungal cell were shifted by the co-cultured metha- Carbamic acid 1.323 0.008 1.396 nogen. The pathways in the cytosol were inhibited and the Cystin 0.105 0.008 1.809 pathways in the hydrogenosome were stimulated, resulting Fructose 0.157 0.008 1.627 in increased provision of H+ for the co-cultured methano- Glutamic acid 1.235 0.008 1.562 gen. The production of lactate from pyruvate in the cytosol Glutamine 1.503 0.008 1.573 decreased in the supernatant by 15%, which is a process of Glycerol-3-phosphate 0.812 0.008 1.593 + Isoleucine 1.209 0.008 1.577 H consumption (from nicotinamide adenine dinucleotide Lactate 0.851 0.008 1.620 to nicotinamide adenosine denucleotide) [8,15,21]. The an- Lauric acid 1.371 0.008 1.527 aerobic fungus does not possess a complete TCA cycle in the Malic acid 0.273 0.008 1.736 cytosol but has reductive (from oxaloacetate to succinate) Myo-inositol 2.534 0.008 1.660 and oxidative (from citrate to α-ketoglutarate) branches of it Myo-inositol-2-phosphate 1.767 0.008 1.372 [22,23]. Present study showed that citrate was accumulated Oxalate 2.195 0.008 1.387 in the fungal cytosol when co-cultured with the methanogen, Proline 1.763 0.008 1.403 which was revealed by the increase of citrate in the fungal Pyruvic acid 2.655 0.008 1.695 cell (7.62-fold). This is consistent with our previous report Threonine 1.293 0.008 1.761 that citrate was accumulated in the co-culture of the anaerobic UN01 2.983 0.008 1.521 Valine 1.387 0.008 1.722 fungus and the methanogen [22]. However, no observed (p Citric acid 0.213 0.012 1.653 = 0.265) increase of α-ketoglutarate in the cytosol of the an- Ethanedioic acid 2.913 0.012 1.473 aerobic fungus co-cultured with the methanogen implies that Glycine 1.245 0.016 1.363 the oxidative branch was not affected by the co-cultured Heptanoic acid 1.132 0.016 1.316 methanogen, except for citrate accumulation. The content 9-Octadecenoic-acid 2.926 0.032 1.212 of fumarate in the fungal cells between two groups was not Fumaric acid 1.269 0.032 1.382 significantly different (p = 1), and even an increase in ma- Glycerol-3TMS 1.180 0.032 1.593 late was observed (3.305-fold in the cell, p = 0.056). L-Asparagine 1.209 0.032 1.419 Previous studies reported significant accumulation of ac- Ua8 0.487 0.032 1.188 etate in the supernatant of the anaerobic fungus co-cultured Uracil 1.266 0.032 1.367 with the methanogen [6-8], which implied that the metabo- bs, supernatant of the co-culture of anaerobic fungus and methanogen; as, supernatant of the anaerobic fungal monoculture; VIP, variable importance plot. lism in the hydrogenosomes of the anaerobic fungus was 1) When p<0.05 and VIP>1, there is a significant difference in metabolites stimulated by the co-cultured methanogen. This was proved between bc and ac.

Figure 5. Effects of the co-cultured methanogen on the metabolic pathway of the anaerobic fungus. Metabolic pathway was drawn based on previous reports by Cheng et al [8], Li et al [15], and Li et al [21]. Red, stimulated pathways; green, inhibited pathways; black, unaffected pathways.

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